Biology subjects

Thuenauer, R.

Publications and source records attributed to Thuenauer, R..

11 recordsLinked to original sources

bioRxiv2026

Intracellular compartmentalization shapes lipid access and metabolic fitness of mycobacteria

Intracellular mycobacteria encounter distinct metabolic environments as they transition between vacuolar and cytosolic compartments within host cells, yet how nutrient access is shaped by this compartmentalization remains poorly understood. Here, we use Mycobacterium marinum fatty acyl- CoA ligase 6 (FACL6) as a functional entry point to examine how lipid acquisition and processing are coordinated during intracellular infection. By combining host and bacterial genetic perturbations with dual RNA-sequencing and high-resolution imaging in genetically tractable amoebal infection models, and validating key phenotypes in mammalian cells, we show that lipid metabolic programs in intracellular mycobacteria are tightly linked to subcellular localization. Sterol utilization and neutral lipid storage are preferentially engaged during the intravacuolar phase, whereas cytosolic exposure is associated with reduced lipid accumulation. Deletion of facl6 disrupts this coordinated scenario, resulting in altered bacterial cell envelope architecture, premature membrane damage, defective neutral lipid storage, and reduced intracellular fitness despite enhanced cytosolic access. Together, these findings reveal that loss of FACL6 causes intrinsic defects in lipid handling and highlight how compartment-specific lipid environments shape the outcome of mycobacterial infection. TeaserLoss of FACL6 reveals how intracellular compartmentalization shapes mycobacterial lipid metabolism

cell biology↗

bioRxiv2025

Clathrin adaptors drive phase separation in endocytosis and trafficking

Liquid-liquid phase separation (LLPS) underlies the formation of biomolecular condensates that organize cellular processes, including endocytosis and membrane trafficking. Although LLPS has been implicated in clathrin-mediated pathways, the specific contribution of adaptor proteins to condensate formation and function remains unclear. Here we show that two yeast adaptors, Ent5 and Sla2, undergo LLPS in vitro and that this property correlates with their distinct roles in membrane trafficking and condensate recruitment in vivo. Ent5 condensation is driven by a dynamic helix within its disordered region that acts as a molecular "sticker." Deletion of this helix disrupts membrane-associated condensation and leads to altered cargo trafficking dynamics in vivo, with Ent5-dependent transport events becoming delayed. In contrast, Sla2 behaves both as a driver and as a client of LLPS, with its coiled-coil (CC) region mediating condensation. Together, these findings reveal that endocytic adaptors can promote condensate formation through distinct structural features, thereby coupling clathrin binding and membrane association through phase separation.

biophysics↗

bioRxiv2025

ChipNMR: Hyperpolarized NMR for non-invasive metabolic flux analysis in perfused microfluidic chips

Dissolution dynamic nuclear polarization NMR Spectroscopy (dDNP-NMR) has become a transformative tool for metabolic studies by significantly enhancing signal sensitivity more than three orders of magnitude compared to traditional NMR. However, NMR detection probes are optimized for round narrow glass tubes typically 5 mm in diameter, which impose constraints on their utility for metabolic studies of adhernt cells. Here, we present a novel NMR probe head integrated with a custom microfluidic chip that facilitates real-time monitoring of hyperpolarized substrate conversion from adhernt cells. This system enables metabolic flux analysis in a controlled, in vitro environment, as demonstrated by tracking the conversion of [1-13C] pyruvate to [1-13C] lactate in HeLa cells over 48 hours. To the best of our knowledge, this is the first demonstration of cell metabolism from an adhering monolayer of mammalian cells in combination with hyperpolarized NMR. The custom microfluidic chip design is modular and adaptable allowing expansion to dual-chamber chips, demonstrating its potential in applications for more complex cellular environments, such as Organ-on-a-Chip systems.

biochemistry↗

bioRxiv2024

Subtleties in Clathrin Heavy Chain Binding Boxes provide selectivity among Adaptor Proteins of Budding Yeast

Clathrin, forming the triskelion network, orchestrates highly regulated cellular processes facilitating cargo internalization and trafficking in eukaryotes, with its N-terminal domain (NTD) pivotal for adaptor protein (AP) interactions. The NTD contains up to four AP-binding sites, and their roles in preferential occupancy by APs have not been addressed. Here, employing a combination of integrative biophysical and structural approaches together with in vivo functional experiments, we investigated the binding hierarchy and selectivity of adaptors for clathrin, aiming to understand the evolutionary conservation of redundant APs and their specialized roles in endocytosis and cellular trafficking mechanisms. We found that yeast epsin Ent5 displayed the highest affinity for clathrin, indicating its significant role in cellular trafficking processes. Epsins Ent1 and Ent2, which are crucial for endocytosis but described to have redundant functions, revealed distinct binding patterns; Ent1 demonstrated stronger interactions with clathrin than Ent2, explaining its functional divergence towards actin binding. Despite both having actin anchoring domains, since Ent1 is actually more stably recruited by clathrin, it would provide a better actin anchoring function. These results offer molecular insights into AP selectivity, suggesting they competitively bind clathrin while also targeting different clathrin sites.

molecular biology↗

bioRxiv2024

Podocyte exopher-formation as a novel pathomechanism in membranous nephropathy

BackgroundMembranous nephropathy (MN) is caused by autoantibody binding to podocyte foot process antigens such as THSD7A and PLA2R1. The mechanisms of the glomerular antigen/autoantibody deposition and clearance are unknown. MethodsWe explore the origin and significance of glomerular accumulations in (1) diagnostic and follow-up biospecimens from THSD7A+ and PLA2R1+-MN patients compared to nephrotic non-MN patients, and (2) in experimental models of THSD7A+-MN. ResultsWe discovered podocyte exophers as correlates of histological antigen/autoantibody aggregates found in the glomerular urinary space of MN patients. Exopher vesicle formation represents a novel form of toxic protein aggregate removal in Caenorhabditis elegans neurons. In MN patients, podocytes released exophers to the urine. Enrichment of exophers from MN patient urines established them as a glomerular exit route for antigens and bound autoantibody. Exophers also carried disease-associated proteins such as complement and provided a molecular imprint of podocyte injury pathways. In experimental THSD7A+-MN, exophers were formed from podocyte processes and cell body. Their formation involved the translocation of antigen/autoantibody from the subepithelial to the urinary side of podocyte plasma membranes. Urinary exopher-release correlated with lower albuminuria and lower glomerular antigen/autoantibody burden. In MN patients the prospective monitoring of urinary exopher abundance and of exopher-bound autoantibodies was additive in the assessment of immunologic MN activity. ConclusionsExopher-formation and release is a novel pathomechanism in MN to remove antigen/autoantibody aggregates from the podocyte. Tracking exopher-release will add a non-invasive diagnostic tool with prognostic potential to clinical diagnostics and follow-up of MN patients.

cell biology↗

bioRxiv2023

Unveiling the cell biology of hippocampal neurons with dendritic axon origin

In mammalian axon-carrying-dendrite (AcD) neurons, the axon emanates from a basal dendrite, instead of the soma, to create a privileged route for action potential generation at the axon initial segment (AIS). However, it is unclear how such unusual morphology is established and whether the structure and function of the AIS in AcD neurons is preserved. Here, we show that the AcD neurons follow an intrinsically encoded developmental program where a single precursor neurite first gives rise to the axon and then to the AcD. The AIS possesses a similar cytoskeletal architecture as the canonical AIS that stems from the soma, and similarly functions as a trafficking barrier to retain axon-specific molecular composition. However, unlike soma-derived AIS, the AIS of AcD neurons does not undergo homeostatic-plasticity, contains less cisternal organelles and receives fewer inhibitory inputs. These distinct features of the AIS could account for the higher intrinsic excitability of AcD neurons. HighlightsO_LIThe development of AcD neurons is intrinsically driven and does not depend on specific connectivity patterns C_LIO_LIAcD neurons generate the axon and AcD from the same precursor neurite C_LIO_LIThe stem dendrite of an AcD neuron displays axon-like microtubule organization C_LIO_LISimilar to the AIS emerging from the soma, the AIS of AcD neurons can selectively filter dendritic cargo C_LIO_LIThe AIS of AcD neurons does not undergo chronic homeostatic plasticity and receives fewer inhibitory inputs C_LI

neuroscience↗

bioRxiv2023

Identifying repurposed drugs with moderate anti-influenza virus activity through computational prioritization of drug-target pairs

Influenza A virus (IAV) causes up to five million cases of severe illness and half a million deaths worldwide each year. While there are a few clinically approved drugs for treating IAV, they are challenged by the rapid evolution of the virus leading to emergence of drug resistance and the adverse effects of the drugs. Targeting host cellular factors that support virus replication could limit resistance, increase the broad-spectrum antiviral properties of drugs, and benefit from repurposing drugs already existing against those factors. However, selecting the right drug-target pairs with low toxicity and minimal adverse effects has been challenging, even though hundreds of cellular host factors have been identified. In this study, we applied a computational and knowledge-based drug-target prioritization approach to identify promising drug-target pairs. We selected five pairs for experimental validation: telmisartan-Angiotensin II receptor, type 1 (AGTR1), metoclopramide hydrochloride-Cholinergic receptor muscarinic 1 (CHRM1), cefepime hydrochloride-phosphogluconate dehydrogenase (PGD), ranolazine dihydrochloride-sodium channel voltage-gated type v alpha subunit (SCN5A), and ofloxacin-topoisomerase II alpha 170kDa (TOP2A). Except for cefepime hydrochloride, all four drugs showed significant plaque reduction in Madin Darby canine kidney (MDCK) cells. In the immunofluorescence assay, metoclopramide hydrochloride, ranolazine dihydrochloride, and telmisartan showed antiviral activity in MDCK and/or adenocarcinoma human alveolar basal epithelial (A549) cell lines. In conclusion, our approach can prioritize and identify drugs with antiviral activity against influenza virus. Refining and strengthening such approaches could be valuable for rapid antiviral discovery and pandemic preparedness. HighlightsO_LIComputational drug-target prioritization indicated drugs for validation C_LIO_LITelmisartan showed anti-influenza virus activity in MDCK cells C_LIO_LIMetoclopramide and ranolazine showed anti-influenza activity in A549 and MDCK cells C_LI

systems biology↗

bioRxiv2023

Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, the sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as tag on the protein of interest and the second fragment as supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells. SynopsisWe present here a novel method to determine the arrival of a membrane protein of interest at the cell surface in real time and with sufficient sensitivity to achieve single-cell resolution. This allows measuring detailed kinetics of plasma membrane trafficking and thus to detect also minor changes in protein sorting and intracellular trafficking. Furthermore, the single-cell sensitivity of the method will enable to systematically characterize variations in trafficking between neighboring cells within a multicellular organism.

cell biology↗

bioRxiv2022

The lectin LecB induces patches with basolateral characteristics at the apical membrane to promote Pseudomonas aeruginosa host cell invasion

The opportunistic bacterium Pseudomonas aeruginosa can infect mucosal tissues of the human body. To persist at the mucosal barrier, this highly adaptable pathogen has evolved many strategies, including invasion of host cells. Here, we show that the P. aeruginosa lectin LecB binds and cross-links fucosylated receptors at the apical plasma membrane of epithelial cells. This triggers a signaling cascade via Src kinases and PI3K leading to the formation of patches enriched with the basolateral marker PIP3 at the apical plasma membrane. This identifies LecB as causative bacterial factor for activating this well-known host cell response that is elicited upon apical binding of P. aeruginosa. Downstream of PI3K, Rac1 is activated to cause actin rearrangement and the outgrowth of protrusions at the apical plasma membrane. LecB-triggered PI3K activation also results in aberrant recruitment of caveolin-1 to the apical domain. In addition, we reveal a positive feedback loop between PI3K activation and apical caveolin-1 recruitment, which provides a mechanistic explanation for the previously observed implication of caveolin-1 in P. aeruginosa host cell invasion. Interestingly, LecB treatment also reversibly removes primary cilia. To directly prove the role of LecB for bacterial uptake, we coated bacteria-sized beads with LecB, which drastically enhanced their endocytosis. Furthermore, LecB deletion and LecB inhibition with L-fucose diminished the invasion efficiency of P. aeruginosa bacteria. Taken together, our study identifies LecB as missing link that can explain how PI3K signaling and caveolin-1 recruitment are triggered to facilitate invasion of epithelial cells from the apical side by P. aeruginosa. ImportanceAn intriguing feature of the bacterium P. aeruginosa is its ability to colonize highly diverse niches. P. aeruginosa can, besides biofilm formation, also enter and proliferate within epithelial host cells. Moreover, research during recent years has shown that P. aeruginosa possesses many different mechanisms to invade host cells. In this study we identify LecB as novel invasion factor. In particular, we show that LecB activates PI3K signaling, which is connected via a positive feedback loop to apical caveolin-1 recruitment, and leads to actin rearrangement at the apical plasma membrane. This provides a unifying explanation for the previously reported implication of PI3K and caveolin-1 in P. aeruginosa host cell invasion. In addition, our study adds a further function to the remarkable repertoire of the lectin LecB, which is all brought about by the capability of LecB to recognize fucosylated glycans on many different niche-specific host cell receptors.

microbiology↗

bioRxiv2019

The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as crosslinker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of {beta}1-integrins on the basolateral plasma membrane of epithelial cells and caused rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive {beta}1-integrins and also of complete 3{beta}1-integrin - laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and caused the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by crosslinking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results we suggest that LecB has a role in disseminating bacteria along the cell - basement membrane interface. ImportancePseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes for nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interaction is not well understood. The significance of our research is in identifying cellular mechanisms how LecB facilitates P. aeruginosa infection: We introduce LecB as new member to the list of bacterial molecules that bind integrins and show that P. aeruginosa can efficiently move forward underneath attached epithelial cells by loosening cell - basement membrane attachment in a LecB-dependent manner.

microbiology↗

bioRxiv2019

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria employ lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection.\n\nHere, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, that extend beyond its role as an adhesion molecule. We found that LecB associates with several growth factor receptors and dampens their signalling pathways, leading to the arrest of cell cycle. Additionally, we describe a novel LecB-triggered mechanism to downregulate host cell receptors by showing that LecB leads to insulin-like growth factor receptor 1 internalisation, without receptor activation, and subsequent missorting towards intracellular endosomal compartments.\n\nOverall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.

cell biology↗